2-(3′-Hydroxypropylidene)-1α-hydroxy-19-norvitamin D compounds with truncated side chains

Our recent studies with 2-(3′-hydroxypropylidene) analogs of 1α,25-dihydroxy-19-norvitamin D₃ showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1α-hydroxy-19-norvitamin D analogs characterized by the presence of a 3′-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8β-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig–Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7–11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1α,25-(OH)₂D₃ (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.     

Selective analogs of 1α,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1α,25-dihydroxyvitamin D₃ synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3′-propylidene-19-nor-(20S or 20R))-1α,25-dihydroxyvitamin D₃ and 2-methylene-19-21-dinor-1α,25-dihydroxyvitamin D₃. Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D₃ (2MD), the 2α-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1α-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1α,25-dihydroxyvitamin D₃. These latter compounds do not elevate serum calcium until doses of the order of >300 μg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D₃ (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1α,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1α,25-dihydroxyvitamin D₃. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1α-hydroxyvitamin D₃ compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by ¹H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1α,25-dihydroxyvitamin D₃ was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ and 22-iodo-1α,25-dihydroxyvitamin D₃ isomers were ca. ten times less potent than the natural hormone 1α,25-(OH)₂D₃ both in intestinal calcium transport and bone calcium mobilization.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Design and synthesis of tetraol derivatives of 1,12-dicarba-closo-dodecaborane as non-secosteroidal vitamin D analogs

Vitamin D receptor (VDR), a nuclear receptor for 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃, 1), is a promising target for multiple clinical applications. We recently developed non-secosteroidal VDR ligands based on a carbon-containing boron cluster, 1,12-dicarba-closo-dodecaborane (p-carborane), and examined the binding of one of them to VDR by means of crystallographic analysis. Here, we utilized that X-ray structure to design novel p-carborane-based tetraol-type vitamin D analogs, and we examined the biological activities of the synthesized compounds. Structure–activity relationship study revealed that introduction of an ω-hydroxyalkoxy functionality enhanced the biological activity, and the configuration of the substituent significantly influenced the potency. Among the synthesized compounds, 4-hydroxybutoxy derivative 9a exhibited the most potent activity, which was equal to that of the secosteroidal vitamin D analog, 19-nor-1α,25-dihydroxyvitamin D₃ (2).     

Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1α,25(OH)₂D₃can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized ³H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1α,25(OH)₂D₃ analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2α- or 2β-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1α,25(OH)₂D₃. The ³H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1α,25(OH)₂D₃, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ or 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ for 7 days. 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ and 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ were also shown to be about 10-fold more active than 1α,25(OH)₂D₃ in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1α,25(OH)₂D₃ molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Ceriopsins A–D, diterpenoids from Ceriops decandra

Chemical examination of the ethyl acetate solubles of the CH₃OH:CH₂Cl₂ (1:1) extract of the roots of Ceriops decandra collected from Kauvery estuary resulted in the isolation of four new diterpenoids, ceriopsins A–D (1–4). The structures of the new diterpenoids were elucidated by a study of their physical and spectral data as methyl 17-hydroxy-16-oxobeyeran-18-oate (1), methyl 16(R)-16,17-dihydroxybeyeran-18-oate (2), 1β,15(S)-isopimar-7-ene-1,15,16-triol (3), and 8,15(R)-epoxypimarane-1β,16-diol (4).     

m-Alkyl, α,α,α-trifluoroacetophenones: A new class of potent transition state analog inhibitors of acetylcholinesterase

A series of m-alkyl α,α,α-trifluoroacetophenones (1–5) was synthesized and evaluated as inhibitors of acetylcholinesterase from Torpedo california. All ketones (1–5) were found to be potent inhibitors of the enzyme; m-t-butyl α,α,α-trifluoroacetophenone (4) was the most potent inhibitor with a K_i value of 3.7 pM.     

Synthesis and glycogen phosphorylase inhibitory activity of N-(β-d-glucopyranosyl)amides possessing 1,4-benzodioxane moiety

A series of N-(β-d-glucopyranosyl)amides 5d–i were synthesized by PMe₃ mediated Staudinger reaction of O-peracetylated β-d-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d–i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure–activity relationships of these compounds are also discussed in this paper.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Synthesis and structure-activity relationship of 1H-indole-3-carboxylic acid pyridine-3-ylamides: a novel series of 5-HT2C receptor antagonists

A novel series of 1H-indole-3-carboxylic acid pyridine-3-ylamides were synthesized and identified to show high affinity and selectivity for 5-HT_2C receptor. Among them, 1H-indole-3-carboxylic acid[6-(2-chloro-pyridin-3-yloxy)-pyridin-3-yl]-amide (15k) exhibits the highest affinity (IC₅₀ = 0.5 nM) with an excellent selectivity (>2000 times) over other serotonin (5-HT_1A, 5-HT_2A, and 5-HT₆) and dopamine (D₂–D₄) receptors.     

Extra-renal 25-hydroxyvitamin D3-1α-hydroxylase in human health and disease

Although ectopic expression of 25-hydroxyvitamin D₃-1α-hydroxylase (1α-OHase) has been recognized for many years, the precise function of this enzyme outside the kidney remains open to debate. Three specific aspects of extra-renal 1α-OHase have attracted most attention: (i) expression and regulation in non-classical tissues during normal physiology; (ii) effects on the immune system and inflammatory disease; (iii) expression and function in tumors. The most well-recognized manifestation of extra-renal 1α-OHase activity remains that found in some patients with granulomatous diseases where locally synthesized 1α,25(OH)₂D₃ has the potential to spill-over into the general circulation. However, immunohistochemistry and mRNA analyses suggest that 1α-OHase is also expressed by a variety of normal human tissues including the gastrointestinal tract, skin, vasculature and placenta. This has promoted the idea that autocrine/paracrine synthesis of 1,25(OH)₂D₃ contributes to normal physiology, particularly in mediating the potent effects of vitamin D on innate (macrophage) and acquired (dendritic cell) immunity. We have assessed the capacity for synthesis of 1,25(OH)₂D₃ in these cells and the functional significance of autocrine responses to 1α-hydroxylase. Data suggest that local synthesis of 1,25(OH)₂D₃ may be a preferred mode of response to antigenic challenge in many tissues.